CN1182795A

CN1182795A - Gene encoding carboxypeptidase of aspergillus niger

Info

Publication number: CN1182795A
Application number: CN97125406A
Authority: CN
Inventors: D·S·亚夫尔; S·A·索姆普森
Original assignee: Novo Nordisk Biotech Inc
Current assignee: Novozymes Inc
Priority date: 1994-09-20
Filing date: 1997-12-03
Publication date: 1998-05-27
Also published as: US5705376A; US5939305A; CN1079831C; DE69533420T2; WO1996009397A1; EP0734444B1; JP3236862B2; FI962099A0; DE69533420D1; CN1137293A; ZA957883B; JPH09501061A; EP0734444A1; US5688663A; ATE274590T1; US5594119A; AU3593195A; US5693510A; FI962099A; KR970700769A

Abstract

The present invention relates to a gene encoding an ascomycete or deuteromycete carboxypeptidase Y gene, and host cells modified so as to produce reduced amounts of carboxypeptidase.

Description

The gene of encoding carboxypeptidase of aspergillus niger

The gene of fungi vacuole protein enzyme the present invention relates to encode.Particularly the present invention relates to thread sac fungi or imperfect fungi fungi, for example the carboxypeptidase gene of Aspergillus.

The fungi vacuole is to contain many lytic enzymes, comprises the acid organ of several proteolytic enzyme, and is equal to mammiferous lysosome basically.Identified and characterized the particularly lytic enzyme of one or more fungies of zymic; These comprise protease A (PEPA or PrA), proteolytic enzyme B (PrB) or aminopeptidase (APE), dipeptidylaminopeptidase B (DPAPB), carboxypeptidase y (CPY) and Carboxypeptidase S (CPS).Most of vacuole lytic enzymes are as nonactive precursor and synthetic glycoprotein.In fact, except that APE, above-mentioned all proteolytic enzyme of mentioning all have and cause the signal peptide of transition by Secretory Pathway.In the golgi body, vacuole protein separates with secretory protein, finally is transported in the vacuole then late.Except that signal peptide, most of vacuole proteins also contain be transported to behind the vacuole cleaved to produce the former peptide of ripe organized enzyme.Verified PrA and the CPY of existing in former peptide is for the vacuole being the required amino acid information of target (Johnson et al., Cell 48:875-885,1987; Rothman etal., PNAS USA 83:3248-3252,1989; Valls et al., Cell 48:887-897; Valls et al.J.Cell Biol.111:361-368,1987).For CPY, shown that it is essential (Valls etal.J.Cell Biol.1l1:361-368,1990) for being positioned vacuole that four amino-acid residues (QRPL) are arranged.In case after being transported to vacuole, the former peptide of protease A (pep4) cracking CPY and PrB is so that activator enzyme (Ammerer et al., Mol.Cell.Biol.6:2490-2499,1986; Woolford et al., Mol, Cell.Biol.6:2500-2510).

In cereuisiae fermentum, three class mutant (Bankaitis et al., PNAS USA 83:9075-9079,1986 of mispointing or misclassification vacuole protein have been identified; Robinson et al., Mol.Cell.Biol., 8:4936-4948,1988; Rothman et al., EMBOJ.8:2057-2065,1989; Rothman and Steven, Cell 47:1041-1051,1986).These mutant are called as vps or vacuole protein classification mutant.Utilize a kind of selection to separate several such mutant, the expression excessively that described selection is based on the height copy vacuole protein enzyme of observing on the plasmid can cause secreting vacuole proteolytic enzyme (Steven etal., J.Cell Biol., 102:1551-1557,1986:Rothman et al, PNAS USA83:3248-324l, 1986).This shows late to have saturated classification mechanism in the golgi body.

In cereuisiae fermentum, also show, disappearance PEP4, the bacterial strain of CPY and PrB is because the heterologous protein of the minimizing production higher level of required product albumen hydrolysis.Therefore, be used to the recombinant production heterologous protein owing to produce the fungi of the vacuole protein enzyme of being studied, thereby from a business perspective, described vacuole protein enzyme is very important.The existence of these proteolytic enzyme is very headachy in fermentation, the required protein because they can also be degraded, thus obviously reduce productivity.Can help eliminating any existing proteinic function by its gene of broken or disappearance coding; But, do the corresponding gene that at first will identify and be separated among the required host like this.As mentioned above, from various yeast strains, only identified described gene seldom; Yet the gene few people of coding vacuole protein enzyme know in thread ascomycetes or imperfect fungi.For example, the coding PEPC (Frederich et al., Gene 125:57-64,1993) and PEPE (Jarai et al., Gene 145:171-178, the 1994) gene of two kinds of aspergillus niger vacuole protein enzymes have in addition been separated.PEPC proteins encoded enzyme B (PrB) homologue, the homologue of PEPE proteins encoded enzyme A.PEP4 gene (Bowman, the 17th funga of the Neutospora Crassa of coding PrA homologue have also been cloned; Genetics Conference, 1993).This paper has described the gene from the coding vacuole CPY of thread ascomycetes or imperfect fungi first.

The present invention relates to contain the nucleic acid construct of thread ascomycetes of coding or imperfect fungi carboxypeptidase y, and recombinant production is by its encoded protein matter.As used herein, " nucleic acid construct " refers to any cDNA, genomic dna, the nucleic acid molecule in synthetic DNA or RNA source.Term " construct " refers to nucleic acid fragment, and described nucleic acid fragment can be strand or two strands, can be completely or partially separated from natural gene, or can be modified to contain to make up and juxtaposed dna fragmentation in the non-existent mode of nature.Described construct can also contain other nucleic acid fragments.In preferred embodiments, the carboxypeptidase of described sequence encoding Aspergillus.The present invention also provides the method for producing the thread ascomycetes or the imperfect fungi cell that do not produce carboxypeptidase, comprise fragmentation or disappearance carboxypeptidase gene preventing the expression of functional enzyme, or classical mutagenesis by utilizing physical or chemical treatment is produced the cell that the CPY ability reduces or do not had to produce it.In addition, the present invention also comprises can not production function carboxypeptidase or with for the amount of wild type strain production, the thread ascomycetes or the imperfect fungi of the carboxypeptidase of production reduction.Described biology provides the basis of improving the recombinant protein production method, and wherein the nucleic acid construct with code for said proteins transforms carboxypeptidase defective type microorganism, cultivates under the described proteinic condition of expression then.Accompanying drawing is described dna sequence dna and the translation thereof that Fig. 1 represents aspergillus niger Bo-1 genome C PY clone.Fig. 2 represents dna sequence dna and the translation thereof of aspergillus niger SFAG2 CPYcDNA.The site of signal peptidase cracked expection and the N-end of ripe CPY have been indicated.Fig. 3 has illustrated used construct in destroying CPY.

Utilize cereuisiae fermentum CPY, little purple mould Carboxypeptidase S l (Svedsen etal., FEBS333:39-43,1993, with Fructus Hordei Germinatus carboxypeptidase-MIII (Sorensen et al., Carlsberg res.Commun.54:193-202,1993), begin to attempt separating the Aspergillus carboxypeptidase y by designing a series of degenerate oligonucleotide.Described oligonucleotide sequence hereinafter is provided among the embodiment.Utilize Aspergillus niger strain Bo-1 genomic dna for the PCR of template reaction in, with these sequences with the various primers that are combined as.Two reactions wherein are (with primer 1-1 and 2-1; And 1-2 and 2-2) produced the amplified production of a 1100bp, with its subclone and order-checking, but there are not subclone and the obvious homology of CPY that is accredited as required gene.Carried out using primer 3-1 then, 3-2,4-1,4-2, other PCR reactions of 2-1 and 2-2.(use primer 4-1 and 2-1 in two reactions therein; And 4-2 and 2-1) obtained the amplified production of a 600bp.With this product subclone, and with 11 subclone order-checkings; 9 subclones wherein are consistent, and with the carboxypeptidase y dna homolog in other sources.Survey the aspergillus niger genomic dna with the insertion fragment in the subclone; Use Bamh I, Hind III and SAlI digestion product are observed the hybridization of a band, show to have single CPY gene in aspergillus niger.In 65 ℃, at 1.5 * SSPE, 1.0%SDS is hybridized in 0.5% skimmed milk and the 200 μ g/ml salmon sperm DNAs.

The aspergillus niger genomic dna storehouse of preparation in EMBL4, use then PCRCPY-deutero-gene fragment ( ³²The P-mark) detection, so that separate full-length gene.In about 28,000 phages, pick 11 male; Wherein 9 are used probe hybridization again behind purifying.The total 5.5HindIII fragment of these clones of great majority is accredited as aspergillus niger CPY gene.With this fragment subclone and order-checking; The fragments sequence of the aminoacid sequence that contains CPY coding region and expection is provided in Fig. 1.

Subsequently, screen cDNA storehouse again from different Aspergillus niger strains.From this storehouse, also identify at least one full-length clone.With this cloning and sequencing and in Fig. 2, described described sequence.Expection all has the CPY precursor sequence of two 557 amino acid longs.Relatively homogenic based on from cereuisiae fermentum, from the CPY of aspergillus niger as if having one 137 or 138 amino acid whose before-former peptide.Described gene contains the intron of 61 base pairs.Comparison shows that of two aspergillus niger sequences, some difference aspect aminoacid sequence, this may reflect that described genomic clone with cDNA is the bacterial strain that separates from different.With comparison shows that of corresponding C PY gene of cereuisiae fermentum and C.albican 65% and 66% consistence is arranged respectively.

The present invention is not limited to use the disclosed sequence of Fig. 1 and 2.At first, the present invention comprises that also production is identical with the aminoacid sequence of description in Fig. 1 or 2, but because of the different nucleotide sequence of genetic code degeneracy.In addition,, and utilize technology described herein, can be easy to identify described varient because of the variation that two bacterial strains mutually of the same race obtain on the different sequence of aminoacid sequence explanation in a kind can be tolerated.Therefore when mentioning " aspergillus niger " in this article, should understand and comprise all described varients.Specifically, the present invention also comprises any varient nucleotide sequence, and by its encoded protein matter, wherein said protein kept with the aminoacid sequence shown in Fig. 1 or 2 at least about 80%, preferred about 85%, the best is at least about the homology of 90-95%, and is keeping the activity of sequence described herein in nature.Useful varient for example comprises in the catalogue of above-mentioned definition, and completed conserved amino acid replaces, and described replacement is the described activity of proteins of remarkably influenced not.So-called conservative the replacement, refer to that the amino acid of same type can be by other aminoacid replacement of the type.For example, can exchange nonpolar aliphatic residue Ala, Val, Leu, and Ile, alkaline residue Lys and Arg, or acidic residues Asp and Glu.Similarly, Ser and Thr can guard replacement each other, and Asn and Gln also can.

In addition, isolating gene provides from other thread ascomycetes or imperfect fungi, aspergillus kind for example, and as aspergillus oryzae, smelly aspergillus, aspergillus japonicus, microorganism Aspergillus aculeatus, or separate homogenic method in the Aspergillus nidulans.The thread ascomycetes of other non-aspergillus comprises fusarium, for example, F.graminearum schw, fusarium oxysporum, fusariun solani machete sickle spore (or corresponding teleomorphs) Neurospora crassa, Trichoderma reesei, Tviridae, T.harzianum T.longibranchiatum, little purple mould, some mould, Penicllium chrysogenum, the Salmonella penicillium camemberti, Lou lattice method Te Shi mould, Humicola insolen, grey humicola lanuginosa thermoidea mutation, H.lanuginosa, Scytalidium thermophilumyceliophthora thermophila and Thielavia terrestris.In Southern hybridization, can make probe, to separate the homologous gene in these other kinds with described gene or based on its oligonucleotide.Specifically, with described probe under low or high rigorous condition (for example, at 42 ℃, 5 * SSPE, 0.3%SDS, 200 μ g/ml shear the also salmon sperm DNA of sex change, be respectively 50,35 or 25% and be used for height, prehybridization and hybridization under the low rigorous condition of neutralization) hybridize with the genome or the cDNA of required kind, after the Southerm trace method of standard, to identify and to separate wherein corresponding C PY gene.With degeneracy probe as herein described and also can obtain CPY specificity product from genomic dna of filamentous fungus or the PCR of article one chain cDNA, then can be with it as corresponding genome of probing pin clone or cDNA.

Gene of the present invention is producing thread ascomycetes, and is for example particularly useful during aspergillar carboxypeptidase-deficient mutants.Can reach this purpose with a lot of methods.In one approach, as described further below, a selected marker is cloned into a CPY gene middle part.With restriction enzyme the destructive fragment is discharged from maternal plasmid.Transform selected host cell with described linearizing dna fragmentation.In described host cell, homology is terminal matches with host chromosome, and homologous recombination has produced chromogene and substituted.Draw together amdS with the marks packets selected that fungal cell host can use, pyrG; ArgB, niaD, sC, and hygB.In addition, but can use two-step approach with two kinds of selective markers.In described a kind of method, but be used in cutting in the CPY pairing once so that contain the CPY gene that blocks and the plasmid of selectable marker gene in the hit restriction enzyme digestion that is incorporated into the CPY seat of conversion process.But make the transformant growth selecting to lose on the substratum of selective marker then, for example if when being labeled as pyrG, substratum can contain 5-fluorootic acid.When recombinating between the CPY gene that blocks of wild-type CPY and importing, but losing of selective marker usually taken place.The CPY gene that should only block with about 50% obtained strains, and other 50% contain wild type gene.At Rothstein, Meth.Enzymol.194,281-301 has described described method in 1991.

The CPY deficient mutants that is produced is useful especially when expressing heterologous protein.In the present invention, " heterologous protein " refers to natural non-existent protein in described host, and native sequences has been carried out the natural protein of modifying, or with behind the recombinant DNA technology modification host, it is expressed in the natural protein that changes has quantitatively taken place.This term comprises that also the expression of its mutant used among the host the natural genetic constitution that does not have or used after processing, the natural protein of the natural component that plays a role in uncommon mode in the host.

As already mentioned, the proteolytic enzyme production of being undertaken by selected host cell can be before it be recovered by degraded product the strict productive rate that has limited desired protein.Therefore the elimination of the CPY that is produced by the host or the minimizing of amount have improved the productive rate of desired protein in fact, and the commercial common obsolete host cell that is used for recombinant protein production of viable commercial can be provided.In preferred embodiments, the CPY deficient cell produces at least 25%, and preferably a little less than at least 50%, and the best be the CPY a little less than at least 70%, compares with the corresponding wild type, can lose whole CPY functions at most.

Can be used for the production of recombinant protein with mutant fungal cell of the present invention with the method identical with wild type strain.This area professional is easy to the variation of the various approach of understanding from specific embodiments as herein described, and described embodiment is improved method according to above-mentioned bacterial strains.With expression vector can the expression activity form required gene.Useful expression vector contains can make described carrier stable integration in the genome of host cell or make carrier independent element that duplicates outside the host cell gene group, and preferably one or more can screen the phenotypic markers of transformed host cells at an easy rate.Described expression vector can also comprise the sequence of the promotor of encoding, ribosome bind site, translation initiation codon and, selectable repressor gene or activator gene.In order to express under the guidance of control sequence, used gene preferably can be operated with described control sequence in suitable reading frame and link to each other according to the present invention.

Expression vector can be any carrier that can utilize recombinant DNA technology easily, and the host cell that it imported is depended in the selection of carrier usually.In the preferred embodiment of the invention, host cell is the bacterial strain of Aspergillus.Therefore carrier can be an autonomously replicationg vector, promptly can be as the outer individual and carrier that exists of karyomit(e), and it duplicates and is independent of chromosome duplication, for example plasmid or a kind of extra-chromosomal element, microchromosome, or artificial chromosome.In addition, described carrier can be when it imports in the host cell, the carrier that can be incorporated into host cell chromosome and duplicate with its karyomit(e) of integrating.

In described carrier, the sequence of required gene should can be operated with suitable promoter sequence and be linked to each other.Described promotor can be any dna sequence dna that transcriptional activity is arranged in selected host cell, and can get own coding and host cell homology or allogenic gene.In order in fungal host, to transcribe, the example of useful promotor be own coding aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartate protease, the neutral α-Dian Fenmei of aspergillus niger, aspergillus niger acid stable alpha-amylase, aspergillus niger or bubble are contained glucoamylase (glaA), Rhizomucor miehei lipase, the aspergillus oryzae Sumizyme MP, the promotor of aspergillus oryzae triose-phosphate isomerase or Aspergillus nidulans acetamidase gene.Preferably TAKA-amylase and glaA promotor.

Expression vector of the present invention can also contain suitable transcription terminator, and can operate the polyadenylic acid sequence that links to each other with the dna sequence dna of code separated source gene sequence in eukaryotic cell.Stop and suitable can the obtaining of polyadenylic acid sequence from the source identical with promotor.Described carrier can also contain the dna sequence dna that can carrier be duplicated in the host cell of being studied.The example of described sequence is plasmid pUC19, pACYC177, pUB110, pE194, the replication orgin of pAMB1 and pIJ702.

Described carrier can also contain selected marker, and for example its product replenishes the gene of the defective in the host cell, or gives antibiotics resistance, penbritin for example, kantlex, the gene of paraxin or tetracyclin resistance.The example of aspergillus selective marker comprises amdS, PYrGmargB, niaD sC, and hygB, the mark of generation hygromycin resistance.Preferred amdS and the pyrG that uses Aspergillus nidulans or aspergillus oryzae in the aspergillus host cell.In addition, for example finish selection by cotransformation by the description among the WO91/17243.

Usually obtain extracellular products after preferably expressing.Therefore described protein contains the proparea that expressed protein can be secreted in the substratum.If desired, described proparea can be that the present invention is proteinic natural, or general replacement of the dna sequence dna in encoded corresponding proparea and with different propareas or signal sequence replacement.For example, described proparea can derive from the glucoamylase or the amylase gene of aspergillus kind, the amylase gene of bacillus specie, the lipase of Rhizomucor miehei or proteinase gene, the gene of cereuisiae fermentum α-factor or Niu Qianyuan rennet-based because of.When the host is the fungal cell, particularly preferably be aspergillus oryzae TAKA amylase, aspergillus niger neutral starch enzyme, the maltogenic amylase of bacillus NCIB 11837, the proparea of bacstearothermophilus α-Dian Fenmei or Bacillus licheniformis proteolytic enzyme.A kind of effective signal sequence is aspergillus oryzae TAKA amylase signal, Rhizomucor miehei aspartate protease signal and Rhizomucormiehei lipase signal.

Be respectively applied for connection DNA construct of the present invention, promotor, terminator and other compositions are inserted into the method that contains in the suitable carrier that duplicates required information then, be this area professional know (referring to for example, Sambrook et al., MolecularCloning, 1989).

Can express any required protokaryon or eukaryotic protein with the CPY deficient mutants, and be preferred for expressing eukaryotic protein.These cells are useful especially to be to be applicable to expression catalase, laccase, phenol oxidase, oxydase, oxydo-reductase, cellulase, zytase, peroxidase, lipase, lytic enzyme, esterase, at (cutinase) proteolytic enzyme and other proteolytic ferments, aminopeptidase, carboxypeptidase, phytase, lyase, polygalacturonase and other hydrolyzed pectins (pectinolytic) enzyme, amylase, glucoamylase, tilactase, tilactase, alpha-glucosidase, beta-glucosidase enzyme, mannosidase, isomerase, sucrase, transferring enzyme, rnase, chitinase and deoxyribonuclease.This area the professional should be appreciated that, term " fungal enzyme " not only comprises natural fungi, and comprises through aminoacid replacement, and disappearance adds or for improving its activity, thermostability, pH tolerance etc. and the fungal enzyme of modified.Also can express heterologous protein on the pharmacology, hormone for example, somatomedin, acceptor etc. with described mutant.

Will the present invention is further illustrated with the following example.Example I. separate aspergillus niger CPY gene A. material and method

I. bacterial strain

Used following biomaterial in the method for Miao Shuing hereinafter.E.coli K802 (ek4-(nrca), mcr B, hsdR2, galD2, GalT22, wupE44, metB1; E.coliSOLP (E14-(mcrA) (mcrCB-hsdSMR-mr ^r) 171, sbcC, recB, recJ, uvrC, umuD::Tn5 (kan ^r), lacgryS96, relA1, thi-1, endA1, λ ^R[F ' proAB lacIqZ Δ M15] Su-, E.coliJM101supE, thi-1, Δ (lac-proAB), [F ' traD36, proAB, lacIqZ Δ M15], E.coli XL-1 Blue rec Al, endAl, gyr A96, thi-1, hsdR17, supE44, relA1, lac[F ' proAB lacIqZ Δ M15, Tn10 (tet ^R)], aspergillus niger Bo-1, aspergillus niger SFAG-2.

The ii.PCR amplification

Carry out PCR with standard technique 45 ℃ of annealing.As template, use following degenerate oligonucleotide with aspergillus niger Bo-1 genomic dna.Primer 1-1 (94-282)-GGIGGICCIGGITGYTC primer 1-2 (94-283)-GGIGGICCIGGITGYAG primer 2-1 (94-284)-CCIAGCCARTTRCADAT primer 2-2 (94-285)-CCYAACCARTTRCADAT primer 3-1 (94-331)-GTIGGITTYTCITAYTCIGG primer 3-2 (94-332)-GTIGGITTYAGYTAYAGYGG primer 4-1 (94-329)-GARTCITAYGCIGGICAYTA primer 2-1 (94-284)-GARAGYTAYGCIGGICAYTA I in above-mentioned primer represents inosine, Y represents C or T, R represents A or G, D represents A, G or T.Iii. subclone PCR product

By the explanation of manufacturers, with TA Cloning test kit (Invitrogen) with PCR product subclone to check order.Iv. in λ Zap II body, cut

By going down to posterity in intestinal bacteria SOLR, the method that provides according to Strategene can be gone into the Zap clone from CPY cDNA and be obtained to contain the plasmid that has carried cDNA insertion pBluescript SK carrier then.Fluorescently-labeled Nucleotide is used in the v.DNA order-checking, utilizes the polymerase cycle order-checking to determine nucleotide sequencing.Go up electrophoresis sequencing reaction product at Applied Biosystems automated DNA sequenator (Model 363 A, 1.20 editions). use following CPY Auele Specific Primer; M13 (-48 ) M13 (-20 ) ( Sanger et al.,J.Mol.Biol.143：163-178 ) ：94-376 TCGCTGCCAGTCTATGATTGA94-377 ACATCAACCGCAACTTCCTCT94-378 TTGCCAATGAGAACGGACTGC94-379 CGCACTTACCACGGACATCAT94-503 CAAGCATCCTCAAACTATCGT94-504 GAGACGCATGAAGGTGAAGTT94-505 GCCGTCCCTCCCTTCCAGCAG94-506 GTGCCGACGGGTTCTCCAAGC94-507 GCAGCGAGGAAGAGCGTTGTC94-510 GGGTCATTCTCGGGGTCATTG94-511 GACCCCGAGAATGACCCTGTT94-512 GTAGGGCTTCATCCAGTCACC94-513 TCTCACCGTTCTCACCAGTAA94-514 TCCCTCCCCAAGAAGCACAAC94-528 AGCGTCTGGGTTACTGGTGAG94-529 AAGATCGGCCAGGTCAAGTCC94-530 GAGACGGTGGTAGGGCTTCAT94-531 AACGTCGGTTACTCTTACAGC94-532 GTGGTCGGGGCGGCGGTTGTG94-533 TGTTTGAAGAAGAGGGTAAGC94-575 CGCTGCTACTTGATTTTTCTA94-576 CTCAGCGCCAACAGCCTCAAT94-577 ACCTGCAGTCCGTTCTTATTG94-634 TGCGATCGATTCATTCTCATC94-635 GGAGTAACCGACATTGACAGG94-636 CCTGTCAATGTCGGTTACTCC94-637 GTCCCATGGCAACTTCACCTT94-646 CTTCTCACCGTTCTCACCAGT94-647 CGAGACTCGAAGAACCCTAAGB.

With aspergillus niger Bo-1 genomic dna is template, uses the CPY specificity degenerate oligonucleotide of various combinations, and primer 1-1,1-2,2-1 and 2-2 (Fig. 1) finish the PCR reaction.All reactions all with a circulation be 95 ℃ 5 minutes, 45 ℃ of 1 minute and 72 ℃ 2 minutes then carry out 25 next circulations, promptly 95 ℃ 1 minute, 45 ℃ of 1 minute and 72 ℃ 2 minutes.With reactant sample (10 μ l) electrophoresis, in two reactions, one is used primer 1-2 and 2-1 on sepharose, and another uses primer 1-2 and 2-2, and the amplified production of about 1100bp accounts for major part.Use supposes do not have intron in these oligonucleotide combinations there in the gene of size at 900bp of the product of expection, and use forward direction and reverse primer are with the also order-checking of amplified production subclone of 1100bp.7 subclones are checked order, still, and none and CPY codon homology.

Use condition same as described above, finish and use various combination of primers 3-1,3-2,4-1,4-2, the PCR of 2-1 and 2-2.Electrophoresis sample on sepharose, in two reactions, one is used primer 4-1 and 2-1, and another uses primer 4-2 and 2-1, and the amplified production of about 600bp accounts for major part.Be 600bp greatly less according to this amplified production of predicting with the homology of other carboxypeptidase.With the amplified production subclone of 600bp, determine the dna sequence dna of 11 subclones with forward direction and reverse primer.9 among 11 clones to have 69% consistence with cereuisiae fermentum, is the codon of aspergillus niger CPY.All 9 all mutually the same, illustrates to have only a carboxypeptidase gene in aspergillus niger.The subclone that will contain 600bp aspergillus niger CPY PCR product is called pDSY17.

Survey the Southerm trace of aspergillus niger Bo-1 genomic dna with insertion of pDSY17.Breach-translation kits radiolabeled probe with Gibco-BRL.Used hybridization conditions is in 60 ℃, at 1.5 * SSPE, and 1%SDS and 0.5% skimmed milk and 200 μ g/ml salmon sperm DNAs.In 65 ℃ with 0.2 * SSC, 1%SDS and 0.1% trisodium phosphate be the trace washed twice, each 15 minutes.At BamHI, in the digestion product of HindIII and SalI, about 10,5.5 and the list of 7kb band respectively with the CPY probe hybridization.

For the full-length gene of separation of C PY, the CPY gene fragment that produces with PCR-(with breach-translation kits mark of Gibco-BRL) is surveyed the genomic library among the EMBL4 of the aspergillus niger Bo-1 that contains 26,000 recombinant chous of having an appointment.Be placed on the filter about 28,000 plaques and detection.From these plates, pick 11 male.Behind the purifying, 9 one-levels clone still with the CPY probe hybridization.DNA isolation from 9 clones is carried out restriction enzyme digestion so that it is characterized.From estriction map, there are 7 to be identical in 9.Other two clones are distinctive.From clone's Southern product, determine that in 98 contain the identical HindIII fragment with about 5.5kb of CPY probe hybridization.Do not contain the segmental clone of identical HindIII and contain big (＞12kb) and the HindIII fragment CPY probe hybridization.The HindIII fragment that subclone has is to carry out dna sequencing.The aminoacid sequence of genomic dna sequence and supposition is shown in Fig. 1.

Rescreen the cDNA storehouse among the λ ZAPII (Stratagene) that selects aspergillus niger SFAG-2.42,000 plaques are put on the filter, use above-mentioned CPY probe in detecting then, under the above-mentioned condition of strictness, 112 such plaques are hybridized.Pick 20 of beginning, screen again then, behind the purifying, 18 still with the CPY probe hybridization.From 4 positive colonies, with cutting method DNA isolation in the body of being furnished with λ ZAP test kit.The plasmid of being rescued with EcoRI digestion, then on sepharose electrophoresis to determine to insert segmental size.As if two clones (2-1 and 3-2) have enough big insertion fragment, can contain the cDNA of total length CPY, and respectively contain and have an appointment 1700 and two EcoRI fragments of 250bp.The size of the full-length cDNA of estimating is about 1600bp.Two cDNA clones (2-2 and 2-4) contain less insertion fragment in addition, and still, they all contain the EcoRI fragment of 250bp.Determined the partial dna sequence of clone 3-2 and 2-2,3-2 contains full-length cDNA, and clone 2-2 is blocked about 200bp at 5 ' end.

Determined the global cDNA sequence (Fig. 2) of two chains.Think the CPY precursor of 557 amino acid longs of cDNA coding.Up to the present, the most nucleoside acid difference between cDNA and the genomic clone all is in the hunting range, and this point is not wondrous, because they are from two different Aspergillus niger strains.CPY with cereuisiae fermentum is arranged as the basis, and aspergillus niger CPY seems to contain signal peptide and propetide, and ripe CPY protein is 419 or 420 amino acid longs.Have an appointment respectively 65% and 66% consistence of the CPY of aspergillus niger CPY and cereuisiae fermentum and C.albicans (Mukhtar et al., Gene 121:173-177,1992).II prepares the CPY deficient mutants

In order to produce the Aspergillus niger strain that has lacked CPY, prepare aspergillus oryzae pyrG gene wherein and be inserted into construct (Fig. 3) in the CPY coding region.About 6.5kb HindIII fragment subclone that will contain almost complete CPY gene and about 6kb gene downstream has wherein destroyed the PstI site in pKS+ (Stratagene) derivative.With the recombinant chou of PstI digestion gained, so that lack the fragment of 815bp from the CPY coding region, adding T4 archaeal dna polymerase is mended with all 4 kinds of dNTP and is put down the outstanding end that produces because of PstI digestion.With the blunt end carrier of gained with by with Hind III digestion, fill and lead up with the Klenow fragment then and the fragment of about 3.8kb blunt end of obtaining is continuous.Wherein contain pyrG with Hind III digestion and insert segmental final construct,, on minimum medium, grow into row filter to obtain being used to transform the linear fragment of aspergillus niger pyrG bacterial strain.Contain destructive CPY gene with Southern trace screening transformant with definite those bacterial strains.With Western engram analysis transformant to seek the CPY in cell, lose.Contain ruinate CPY in case identified a bacterial strain, just can determine influence heterologous protein.The preservation of biomaterial

On September 13rd, 1994 following biomaterial is deposited in AgriculturalResearch Service Culture Collection (NRRL) 1815 North UniversityStreet, Peoria, Illinois 61604.The clone preserving number contains the NRRL B-21326E.coli (EMCC#0120) of pDSY23

Sequence table (1) physical data:

(i) applicant:

(A) title: Novo Nordisk Biotech, Inc.

(B) street: 1445 Drew Avenue

(C) city: Davis

(D) state: California

(E) country: US

(F) postcode: 95616-4880

(G) phone: (916) 757-8100

(H) fax: (916) 758-0317

(ii) invention exercise question: the gene of encoding carboxypeptidase of aspergillus niger

(iii) sequence number: 4

(iv) relative address:

(A) addressee: Novo Nordisk of North America, Inc.

(B) street: 405 Lexington Avenue, Suite 6400

(C) city: New York

(D) state: New York

(E) country: U.S.A.

(F) postcode: 10174-6401

(v) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM PL

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release #1.0, Version #1.25 (EPO)

(vi) present application materials:

(A) application number: US

(B) applying date: the 19-9 month-1995

(C) classification number:

(vii) application materials formerly:

(A) application number: US08/309,341

(B) applying date: the 20-9 month-1994

(viii) proxy/act on behalf of data:

(A) name: Lowney, Karen A.

(B) registration number: 31,274

(C) document number: 4247.204-WO

(ix) communications data:

(A) phone: 212 867 0123

(B) fax: the data of 212 867 0298 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 2068 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity is molecule type (ii): genomic dna (vi) originate:

(A) biology: aspergillus niger (ix) feature:

(A) title/keyword: intron

(B) position: 572...632 (ix) feature:

(A) title/keyword: CDS

(B) position: connect (571..633) (xi) sequence description: SEQ ID NO:1:TCCTCTGCCT ACTCATCCCA TCACCATCTC AATTCATACC GCCCCCGTGG GGTTTCAGCA 60CCA ATG AGA GTC CTT CCA GCT GCT ATG CTG GTT GGA GCG GCC ACG GCG 108

Met?Arg?Val?Leu?Pro?Ala?Ala?Met?Leu?Val?Gly?Ala?Ala?Thr?Ala

1 5 10 15GCC?GTT?CCT?CCC?TTC?CAG?CAG?GTC?CTT?GGA?GGT?AAC?GGT?GCC?AAG?CAC 156Ala?Val?Pro?Pro?Phe?Gln?Gln?Val?Leu?Gly?Gly?Asn?Gly?Ala?Lys?His

20 25 30GGT?GCC?GAC?CAT?GCG?GCC?GAG?GTC?CCT?GCG?GAT?CAC?AGT?GCC?GAC?GGG 204Gly?Ala?Asp?His?Ala?Ala?Glu?Val?Pro?Ala?Asp?His?Ser?Ala?Asp?Gly

35 40 45TTC?TCC?AAG?CCG?CTG?CAC?GCA?TTC?CAG?GAG?GAG?CTG?AAG?TCT?CTC?TCT 252Phe?Ser?Lys?Pro?Leu?His?Ala?Phe?Gln?Glu?Glu?Leu?Lys?Ser?Leu?Ser

50 55 60GAC?GAG?GCT?CGT?AAG?CTT?TGG?GAT?GAG?GTG?GCC?AGC?TTC?TTC?CCG?GAG 300Asp?Glu?Ala?Arg?Lys?Leu?Trp?Asp?Glu?Val?Ala?Ser?Phe?Phe?Pro?Glu

65 70 75AGC?ATG?GAT?CAG?AAC?CCT?CTC?TTT?TCC?CTC?CCC?AAG?AAG?CAC?AAC?CGC 348Ser?Met?Asp?Gln?Asn?Pro?Leu?Phe?Ser?Leu?Pro?Lys?Lys?His?Asn?Arg80 85 90 95CGT?CCC?GAC?TCG?CAC?TGG?GAC?CAC?ATC?GTC?CGC?GGC?TCC?GAC?GTT?CAG 396Arg?Pro?Asp?Ser?His?Trp?Asp?His?Ile?Val?Arg?Gly?Ser?Asp?Val?Gln

100 105 110AGC?GTC?TGG?GTC?ACT?GGT?GAG?AAC?GGT?GAG?AAG?GAG?CCC?GAG?GTC?GAT 444Ser?Val?Trp?Val?Thr?Gly?Glu?Asn?Gly?Glu?Lys?Glu?Arg?Glu?Val?Asp

115 120 125GGC?AAG?CTG?GAA?GCC?TAT?GAT?CTC?AGG?GTC?AAG?AAG?ACC?GAT?CCT?GGC 492Gly?Lys?Leu?Glu?Ala?Tyr?Asp?Leu?Arg?Val?Lys?Lys?Thr?Asp?Pro?Gly

130 135 140TCT?CTT?GGC?ATC?GAC?CCC?GGC?GTG?AAG?CAG?TAC?ACC?GGT?TAT?CTC?GAT 540Ser?Leu?Gly?Ile?Asp?Pro?Gly?Val?Lys?Gln?Tyr?Thr?Gly?Tyr?Leu?Asp

145 150 155GAC?AAC?GAG?AAT?GAT?AAG?CAT?TTG?TTC?TAC?GTAAGCACAC?CTTGGTTCAA 590Asp?Asn?Glu?Asn?Asp?Lys?His?Leu?Phe?Tyr160 165GATCACGCTT?TTTATATGCT?CTGGATATCT?AACGCAACTT?AG?TGG?TTC?TTC?GAG 644

Trp?Phe?Phe?Glu

170TCT?CGC?AAT?GAC?CCC?GAG?AAT?GAT?CCC?GTT?GTT?CTG?TGG?CTG?AAC?GGT 692Ser?Arg?Asn?Asp?Pro?Glu?Asn?Asp?Pro?Val?Val?Leu?Trp?Leu?Asn?Gly

175 180 185GGC?CCT?GGG?TGC?TCT?TCC?CTC?ACC?GGT?CTC?TTC?ATG?GAG?CTT?GGC?CCT 740Gly?Pro?Gly?Cys?Ser?Ser?Leu?Thr?Gly?Leu?Phe?Met?Glu?Leu?Gly?Pro190 195 200 205AGC?AGC?ATC?AAC?AAG?AAG?ATC?CAG?CCG?GTC?TAC?AAT?GAC?TAC?GCT?TGG 788Ser?Ser?Ile?Asn?Lys?Lys?Ile?Gln?Pro?Val?Tyr?Asn?Asp?Tyr?Ala?Trp

210 215 220AAC?TCC?AAC?GCG?TCC?GTG?ATC?TTC?CTT?GAC?CAG?CCT?GTC?AAT?GTC?GGT 836Asn?Ser?Asn?Ala?Ser?Val?Ile?Phe?Leu?Asp?Gln?Pro?Val?Asn?Val?Gly

225 230 235TAC?TCC?TAC?AGT?AAC?TCT?GCT?GTC?AGC?GAC?ACG?GTC?GCT?GCT?GGC?AAG 884Tyr?Ser?Tyr?Ser?Asn?Ser?Ala?Val?Ser?Asp?Thr?Val?Ala?Ala?Gly?Lys

240 245 250GAC?GTC?TAT?GCC?TTG?CTT?ACC?CTC?TTC?TTC?AAA?CAA?TTC?CCC?GAG?TAT 932Asp?Val?Tyr?Ala?Leu?Leu?Thr?Leu?Phe?Phe?Lys?Gln?Phe?Pro?Glu?Tyr

255 260 265GCT?AAG?CAG?GAC?TTC?CAC?ATT?GCC?GGT?GAA?TCT?TAT?GCT?GGT?CAC?TAT 980Ala?Lys?Gln?Asp?Phe?His?Ile?Ala?Gly?Glu?Ser?Tyr?Ala?Gly?His?Tyr270 275 280 285ATC?CCC?GTC?TTC?GCT?TCG?GAG?ATC?CTG?TCT?CAC?AAG?AAG?CGC?AAC?ATC 1028Ile?Pro?Val?Phe?Ala?Ser?Glu?Ile?Leu?Ser?His?Lys?Lys?Arg?Asn?Ile

290 295 300AAC?CTG?CAG?TCC?GTT?CTC?ATT?GGC?AAC?GGT?CTC?ACC?GAC?GGA?TAC?ACC 1076Asn?Leu?Gln?Ser?Val?Leu?Ile?Gly?Asn?Gly?Leu?Thr?Asp?Gly?Tyr?Thr

305 310 315CAG?TAC?GAG?TAC?TAC?CGT?CCC?ATG?GCC?TGC?GGT?GAC?GGC?GGT?TAC?CCA 1124Gln?Tyr?Glu?Tyr?Tyr?Arg?Pro?Met?Ala?Cys?Gly?Asp?Gly?Gly?Tyr?Pro

320 325 330GCT?GTC?TTG?GAC?GAG?AGC?TCC?TGC?CAG?TCC?ATG?GAC?AAC?GCT?CTT?CCT 1172Ala?Val?Leu?Asp?Glu?Ser?Ser?Cys?Gln?Ser?Met?Asp?Asn?Ala?Leu?Pro

335 340 345CGC?TGC?CAG?TCT?ATG?ATT?GAG?TCT?TGC?TAC?AGT?TCC?GAG?AGC?GCT?TGG 1220Arg?Cys?Gln?Ser?Met?Ile?Glu?Ser?Cys?Tyr?Ser?Ser?Glu?Ser?Ala?Trp350 355 360 365GTT?TGT?GTC?CCG?GCC?TCC?ATC?TAC?TGT?AAC?AAC?GCC?CTC?CTT?GCC?CCT 1268Val?Cys?Val?Pro?Ala?Ser?Ile?Tyr?Cys?Asn?Asn?Ala?Leu?Leu?Ala?Pro

370 375 380TAC?CAG?CGC?ACT?GGG?CAG?AAC?GTC?TAT?GAT?GTC?CGT?GGT?AAG?TGC?GAG 1316Tyr?Gln?Arg?Thr?Gly?Gln?Asn?Val?Tyr?Asp?Val?Arg?Gly?Lys?Cys?Glu

385 390 395GAT?AGC?TCT?AAC?CTT?TGC?TAC?TCG?GCT?ATG?GGC?TAC?GTC?AGC?GAC?TAC 1364Asp?Ser?Ser?Asn?Leu?Cys?Tyr?Ser?Ala?Met?Gly?Tyr?Val?Ser?Asp?Tyr

400 405 410CTG?AAC?AAG?CCC?GAA?GTC?ATC?GAG?GCT?GTT?GGC?GCT?GAG?GTC?AAC?GGC 1412Leu?Asn?Lys?Pro?Glu?Val?Ile?Glu?Ala?Val?Gly?Ala?Glu?Val?Asn?Gly

415 420 425TAC?GAC?TCG?TGC?AAC?TTT?GAC?ATC?AAC?CGC?AAC?TTC?CTC?TTC?CAC?GGT 1460Tyr?Asp?Ser?Cys?Asn?Phe?Asp?Ile?Asn?Arg?Asn?Phe?Leu?Phe?His?Gly430 435 440 445GAC?TGG?ATG?AAG?CCC?TAC?CAC?CGC?CTC?GTT?CCG?GGA?CTC?CTG?GAG?CAG 1508Asp?Trp?Met?Lys?Pro?Tyr?His?Arg?Leu?Val?Pro?Gly?Leu?Leu?Glu?Gln

450 455 460ATC?CCT?GTC?TTG?ATC?TAT?GCC?GGT?GAT?GCT?GAT?TTC?ATT?TGC?AAC?TGG 1556Ile?Pro?Val?Leu?Ile?Tyr?Ala?Gly?Asp?Ala?Asp?Phe?Ile?Cys?Asn?Trp

465 470 475CTG?GGC?AAC?AAG?GCC?TGG?ACT?GAA?GCC?CTG?GAG?TGG?CCC?GGA?CAG?GCT 1604Leu?Gly?Asn?Lys?Ala?Trp?Thr?Glu?Ala?Leu?Glu?Trp?Pro?Gly?Gln?Ala

480 485 490GAA?TAT?GCC?TCC?GCT?GAG?CTG?GAG?GAT?CTG?GTC?ATT?GTC?GAC?AAT?GAG 1652Glu?Tyr?Ala?Ser?Ala?Glu?Leu?Glu?Asp?Leu?Val?Ile?Val?Asp?Asn?Glu

495 500 505CAC?ACG?GGC?AAG?AAG?ATT?GGC?CAG?GTT?AAG?TCC?CAT?GGC?AAC?TTC?ACC 1700His?Thr?Gly?Lys?Lys?Ile?Gly?Gln?Val?Lys?Ser?His?Gly?Asn?Phe?Thr510 515 520 525TTC?ATG?CGT?CTC?TAT?GGT?GGT?GGC?CAC?ATG?GTC?CCG?ATG?GAC?CAG?CCC 1748Phe?Met?Arg?Leu?Tyr?Gly?Gly?Gly?His?Met?Val?Pro?Met?Asp?Gln?Pro

530 535 540GAG?TCG?AGT?CTC?GAG?TTC?TTC?AAC?CGC?TGG?TTG?GGA?GGT?GAA?TGG?TTC 1796Glu?Ser?Ser?Leu?Glu?Phe?Phe?Asn?Arg?Trp?Leu?Gly?Gly?Glu?Trp?Phe

The data of 545 550 555TAA AGACGTGCTA CCACCGCATA TAGACTTTCT GGTCATTTCG GTGACACTGC 1849AGATATGTTT CTTAACGATA GTTTGAGCAT GCTTGTCAAT GCCCACTAGT CCCGATCCTT 1909ATATGTTGCA TGGTATCTAT GAGTTTTGTC ACTATAGTGC ATTATACATG TGTACTTCGT 1969ATGAGAATGA ATCGATCGCA TTTACACGCA TATAAATAGT ACCCACCTCC GCCTGGACAT 2029GAATTAGGCC CGGCCAGTCG TTTACATACA GTGCTAGAA, 2068 (2) SEQ ID NO:2:

(i) sequence signature:

(A) length: 557 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): protein (vi) originate:

(A) biology: aspergillus niger (xi) sequence description: SEQ ID NO:2:Met Arg Val Leu Pro Ala Ala Met Leu Val Gly Ala Ala Thr Ala Ala1 5 10 15Val Pro Pro Phe Gln Gln Val Leu Gly Gly Asn Gly Ala Lys His Gly

20 25 30Ala?Asp?His?Ala?Ala?Glu?Val?Pro?Ala?Asp?His?Ser?Ala?Asp?Gly?Phe

35 40 45Ser?Lys?Pro?Leu?His?Ala?Phe?Gln?Glu?Glu?Leu?Lys?Ser?Leu?Ser?Asp

50 55 60Glu?Ala?Arg?Lys?Leu?Trp?Asp?Glu?Val?Ala?Ser?Phe?Phe?Pro?Glu?Ser65 70 75 80Met?Asp?Gln?Asn?Pro?Leu?Phe?Ser?Leu?Pro?Lys?Lys?His?Asn?Arg?Arg

85 90 95Pro?Asp?Ser?His?Trp?Asp?His?Ile?Val?Arg?Gly?Ser?Asp?Val?Gln?Ser

100 105 110Val?Trp?Val?Thr?Gly?Glu?Asn?Gly?Glu?Lys?Glu?Arg?Glu?Val?Asp?Gly

115 120 125Lys?Leu?Glu?Ala?Tyr?Asp?Leu?Arg?Val?Lys?Lys?Thr?Asp?Pro?Gly?Ser

130 135 140Leu?Gly?Ile?Asp?Pro?Gly?Val?Lys?Gln?Tyr?Thr?Gly?Tyr?Leu?Asp?Asp145 150 155 160Asn?Glu?Asn?Asp?Lys?His?Leu?Phe?Tyr?Trp?Phe?Phe?Glu?Ser?Arg?Asn

165 170 175Asp?Pro?Glu?Asn?Asp?Pro?Val?Val?Leu?Trp?Leu?Asn?Gly?Gly?Pro?Gly

180 185 190Cys?Ser?Ser?Leu?Thr?Gly?Leu?Phe?Met?Glu?Leu?Gly?Pro?Ser?Ser?Ile

195 200 205Asn?Lys?Lys?Ile?Gln?Pro?Val?Tyr?Asn?Asp?Tyr?Ala?Trp?Asn?Ser?Asn

210 215 220Ala?Ser?Val?Ile?Phe?Leu?Asp?Gln?Pro?Val?Asn?Val?Gly?Tyr?Ser?Tyr225 230 235 240Ser?Asn?Ser?Ala?Val?Ser?Asp?Thr?Val?Ala?Ala?Gly?Lys?Asp?Val?Tyr

245 250 255Ala?Leu?Leu?Thr?Leu?Phe?Phe?Lys?Gln?Phe?Pro?Glu?Tyr?Ala?Lys?Gln

260 265 270Asp?Phe?His?Ile?Ala?Gly?Glu?Ser?Tyr?Ala?Gly?His?Tyr?Ile?Pro?Val

275 280 285Phe?Ala?Ser?Glu?Ile?Leu?Ser?His?Lys?Lys?Arg?Asn?Ile?Asn?Leu?Gln

290 295 300Ser?Val?Leu?Ile?Gly?Asn?Gly?Leu?Thr?Asp?Gly?Tyr?Thr?Gln?Tyr?Glu305 310 315 320Tyr?Tyr?Arg?Pro?Met?Ala?Cys?Gly?Asp?Gly?Gly?Tyr?Pro?Ala?Val?Leu

325 330 335Asp?Glu?Ser?Ser?Cys?Gln?Ser?Met?Asp?Asn?Ala?Leu?Pro?Arg?Cys?Gln

340 345 350Ser?Met?Ile?Glu?Ser?Cys?Tyr?Ser?Ser?Glu?Ser?Ala?Trp?Val?Cys?Val

355 360 365Pro?Ala?Ser?Ile?Tyr?Cys?Asn?Asn?Ala?Leu?Leu?Ala?Pro?Tyr?Gln?Arg

370 375 380Thr?Gly?Gln?Asn?Val?Tyr?Asp?Val?Arg?Gly?Lys?Cys?Glu?Asp?Ser?Ser385 390 395 400Asn?Leu?Cys?Tyr?Ser?Ala?Met?Gly?Tyr?Val?Ser?Asp?Tyr?Leu?Asn?Lys

405 410 415Pro?Glu?Val?Ile?Glu?Ala?Val?Gly?Ala?Glu?Val?Asn?Gly?Tyr?Asp?Ser

420 425 430Cys?Asn?Phe?Asp?Ile?Asn?Arg?Asn?Phe?Leu?Phe?His?Gly?Asp?Trp?Met

435 440 445Lys?Pro?Tyr?His?Arg?Leu?Val?Pro?Gly?Leu?Leu?Glu?Gln?Ile?Pro?Val

450 455 460Leu?Ile?Tyr?Ala?Gly?Asp?Ala?Asp?Phe?Ile?Cys?Asn?Trp?Leu?Gly?Asn465 470 475 480Lys?Ala?Trp?Thr?Glu?Ala?Leu?Glu?Trp?Pro?Gly?Gln?Ala?Glu?Tyr?Ala

485 490 495Ser?Ala?Glu?Leu?Glu?Asp?Leu?Val?Ile?Val?Asp?Asn?Glu?His?Thr?Gly

500 505 510Lys?Lys?Ile?Gly?Gln?Val?Lys?Ser?His?Gly?Asn?Phe?Thr?Phe?Met?Arg

515 520 525Leu?Tyr?Gly?Gly?Gly?His?Met?Val?Pro?Met?Asp?Gln?Pro?Glu?Ser?Ser

The data of 530 535 540Leu Glu Phe Phe Asn Arg Trp Leu Gly Gly Glu Trp Phe545,550 555 (2) SEQ ID NO:3:

(i) sequence signature:

(A) length: 2002 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: linearity is molecule type (ii): cDNA (vi) originate:

(A) biology: aspergillus niger (ix) feature:

(A) title/keyword: intron

(B) position: 349...411 (ix) feature:

(A) title/keyword: CDS

(B) position: connect (348..412) (xi) sequence description: SEQ ID NO:3:GCGGCCGCTG CTACTTGCTT TTTCTAATTT GATACTTTTG TGTCCGTACC GTACCTTCCA 60GACCGCAAGG TACCCATCCT CTACCTACTC ATCCCATCAT CATCTCGATT TCATACCAAC 120CCCGTTGGGT TTCAACACA ATG AGA GTT CTT CCA GCT GCT ATG CTG GTT GGA 172

Met?Arg?Val?Leu?Pro?Ala?Ala?Met?Leu?Val?Gly

1 5 10GCG?GGC?ACT?GCG?GCC?GTC?CCT?CCC?TTC?CAG?CAG?GTC?CTT?GGA?GGT?AAC 220Ala?Gly?Thr?Ala?Ala?Val?Pro?Pro?Phe?Gln?Gln?Val?Leu?Gly?Gly?Asn

15 20 25GGT?GCC?AAG?CAC?GGT?GCC?GAC?CAT?GCG?GCC?GAG?GTC?CCT?GCG?GAT?CAC 268Gly?Ala?Lys?His?Gly?Ala?Asp?His?Ala?Ala?Glu?Val?Pro?Ala?Asp?His

30 35 40AGT?GCC?GAC?GGG?TTC?TCC?AAG?CCG?CTG?CAC?GCA?TTC?CAG?GAG?GAG?CTG 316Ser?Ala?Asp?Gly?Phe?Ser?Lys?Pro?Leu?His?Ala?Phe?Gln?Glu?Glu?Leu

45 50 55AAG?TCT?CTC?TCT?GAT?GAG?GCT?CGT?AAG?CTC?TGG?GAT?GAG?GTT?GCT?AGC 364Lys?Ser?Leu?Ser?Asp?Glu?Ala?Arg?Lys?Leu?Trp?Asp?Glu?Val?Ala?Ser60 65 70 75TTC?TTC?CCG?GAG?AGC?ATG?GAT?CAG?AAC?CCT?CTC?TTC?TCC?CTC?CCC?AAG 412Phe?Phe?Pro?Glu?Ser?Met?Asp?Gln?Asn?Pro?Leu?Phe?Ser?Leu?Pro?Lys

80 85 90AAG?CAC?AAC?CGC?CGC?CCC?GAC?CAC?CAC?TGG?GAC?CAC?ATC?GTC?CGC?GGC 460Lys?His?Asn?Arg?Arg?Pro?Asp?His?His?Trp?Asp?His?Ile?Val?Arg?Gly

95 100 105TCC?GAC?GTT?CAG?AGC?GTC?TGG?GTT?ACT?GGT?GAG?AAC?GGT?GAG?AAG?GAG 508Ser?Asp?Val?Gln?Ser?Val?Trp?Val?Thr?Gly?Glu?Asn?Gly?Glu?Lys?Glu

110 115 120CGT?GAG?GTC?GAT?GGC?AAG?CTG?GAA?GCC?TAT?GAT?CTC?AGG?GTC?AAG?AAG 556Arg?Glu?Val?Asp?Gly?Lys?Leu?Glu?Ala?Tyr?Asp?Leu?Arg?Val?Lys?Lys

125 130 135ACC?GAT?CCT?AGC?TCT?CTT?GGC?ATC?GAC?CCT?GGC?GTA?AAG?CAG?TAC?ACC 604Thr?Asp?Pro?Ser?Ser?Leu?Gly?Ile?Asp?Pro?Gly?Val?Lys?Gln?Tyr?Thr140 145 150 155GGT?TAT?CTC?GAT?GAC?AAC?GAG?AAC?GAC?AAG?CAT?CTG?TTC?TAC?TGG?TTC 652Gly?Tyr?Leu?Asp?Asp?Asn?Glu?Asn?Asp?Lys?His?Leu?Phe?Tyr?Trp?Phe

160 165 170TTC?GAG?TCT?CGC?AAT?GAC?CCC?GAG?AAT?GAC?CCT?GTT?GTT?CTG?TGG?CTG 700Phe?Glu?Ser?Arg?Asn?Asp?Pro?Glu?Asn?Asp?Pro?Val?Val?Leu?Trp?Leu

175 180 185AAC?GGT?GGC?CCT?GGA?TGC?TCT?TCC?CTC?ACC?GGT?CTT?TTC?ATG?GAG?CTC 748Asn?Gly?Gly?Pro?Gly?Cys?Ser?Ser?Leu?Thr?Gly?Leu?Phe?Met?Glu?Leu

190 195 200GGC?CCT?AGC?AGC?ATC?AAC?AAG?AAG?ATC?CAG?CCG?GTC?TAC?AAC?GAC?TAC 796Gly?Pro?Ser?Ser?Ile?Asn?Lys?Lys?Ile?Gln?Pro?Val?Tyr?Asn?Asp?Tyr

205 210 215GCT?TGG?AAC?TCC?AAC?GCG?TCC?GTG?ATC?TTC?CTT?GAC?CAG?CCT?GTC?AAC 844Ala?Trp?Asn?Ser?Asn?Ala?Ser?Val?Ile?Phe?Leu?Asp?Gln?Pro?Val?Asn220 225 230 235GTC?GGT?TAC?TCT?TAC?AGC?AAC?TCT?GCT?GTC?AGC?GAC?ACC?GTT?GCT?GCT 892Val?Gly?Tyr?Ser?Tyr?Ser?Asn?Ser?Ala?Val?Ser?Asp?Thr?Val?Ala?Ala

240 245 250GGC?AAG?GAC?GTC?TAT?GCC?TTG?CTT?ACC?CTC?TTC?TTC?AAA?CAA?TTC?CCC 940Gly?Lys?Asp?Val?Tyr?Ala?Leu?Leu?Thr?Leu?Phe?Phe?Lys?Gln?Phe?Pro

255 260 265GAG?TAT?GCC?AAG?CAG?GAC?TTC?CAC?ATT?GCC?GGT?GAA?TCC?TAT?GCT?GGT 988Glu?Tyr?Ala?Lys?Gln?Asp?Phe?His?Ile?Ala?Gly?Glu?Ser?Tyr?Ala?Gly

270 275 280CAC?TAT?ATC?CCC?GTC?TTT?GCT?TCG?GAG?ATT?TTG?TCT?CAC?AAG?AAG?CGC 1036His?Tyr?Ile?Pro?Val?Phe?Ala?Ser?Glu?Ile?Leu?Ser?His?Lys?Lys?Arg

285 290 295AAC?ATC?AAC?CTG?CAG?TCC?GTT?CTT?ATT?GGC?AAC?GGT?CTC?ACC?GAC?GGT 1084Asn?Ile?Asn?Leu?Gln?Ser?Val?Leu?Ile?Gly?Asn?Gly?Leu?Thr?Asp?Gly300 305 310 315CTC?ACT?CAG?TAC?GAG?TAC?TAC?CGT?CCC?ATG?GCC?TGT?GGT?GAC?GGT?GGT 1132Leu?Thr?Gln?Tyr?Glu?Tyr?Tyr?Arg?Pro?Met?Ala?Cys?Gly?Asp?Gly?Gly

320 325 330TAC?CCA?GCT?GTC?TTG?GAC?GAG?GGC?TCC?TGC?CAG?GCC?ATG?GAC?AAC?GCC 1180Tyr?Pro?Ala?Val?Leu?Asp?Glu?Gly?Ser?Cys?Gln?Ala?Met?Asp?Asn?Ala

335 340 345CTT?CCT?CGC?TGC?CAG?TCT?ATG?ATT?GAG?TCT?TGC?TAT?AGT?TCC?GAG?AGC 1228Leu?Pro?Arg?Cys?Gln?Ser?Met?Ile?Glu?Ser?Cys?Tyr?Ser?Ser?Glu?Ser

350 355 360GCT?TGG?GTT?TGT?GTC?CCG?GCC?TCC?ATC?TAC?TGT?AAC?AAC?GCC?CTC?CTT 1276Ala?Trp?Val?Cys?Val?Pro?Ala?Ser?Ile?Tyr?Cys?Asn?Asn?Ala?Leu?Leu

365 370 375GCC?CCT?TAC?CAG?CGC?ACC?GGA?GAG?AAC?GTC?TAC?GAT?GTT?CGT?GGT?AAG 1324Ala?Pro?Tyr?Gln?Arg?Thr?Gly?Gln?Asn?Val?Tyr?Asp?Val?Arg?Gly?Lys380 385 390 395TGC?GAG?GAT?AGC?TCC?AAC?CTC?TGC?TAC?TCG?GCC?ATG?GGC?TAC?GTC?AGC 1372Cys?Glu?Asp?Ser?Ser?Asn?Leu?Cys?Tyr?Ser?Ala?Met?Gly?Tyr?Val?Ser

400 405 410GAC?TAC?CTG?AAC?AAG?ACC?GAG?GTC?ATT?GAG?GCT?GTT?GGC?GCT?GAG?GTC 1420Asp?Tyr?Leu?Asn?Lys?Thr?Glu?Val?Ile?Glu?Ala?Val?Gly?Ala?Glu?Val

415 420 425AAC?GGC?TAC?GAC?TCG?TGC?AAC?TTT?GAC?ATC?AAC?CGC?AAC?TTC?CTC?TTC 1468Asn?Gly?Tyr?Asp?Ser?Cys?Asn?Phe?Asp?Ile?Asn?Arg?Asn?Phe?Leu?Phe

430 435 440CAC?GGT?GAC?TGG?ATG?AAG?CCC?TAC?CAC?CGT?CTC?GTT?CCG?GGA?CTC?CTG 1516His?Gly?Asp?Trp?Met?Lys?Pro?Tyr?His?Arg?Leu?Val?Pro?Gly?Leu?Leu

445 450 455GAG?CAG?ATC?CCT?GTC?CTG?ATC?TAC?GCT?GGT?GAC?GCC?GAT?TTC?ATC?TGC 1564Glu?Gln?Ile?Pro?Val?Leu?Ile?Tyr?Ala?Gly?Asp?Ala?Asp?Phe?Ile?Cys460 465 470 475AAC?TGG?CTG?GGC?AAC?AAG?GCC?TGG?ACT?GAA?GCC?CTT?GAG?TGG?CCC?GGA 1612Asn?Trp?Leu?Gly?Asn?Lys?Ala?Trp?Thr?Glu?Ala?Leu?Glu?Trp?Pro?Gly

480 485 490CAG?GCT?GAA?TAT?GCC?TCC?GCT?AAG?CTG?GAG?GAC?CTG?GTC?GTG?GTC?GAG 1660Gln?Ala?Glu?Tyr?Ala?Ser?Ala?Lys?Leu?Glu?Asp?Leu?Val?Val?Val?Glu

495 500 505AAT?GAG?CAC?AAG?GGC?AAG?AAG?ATC?GGC?CAG?GTC?AAG?TCC?CAT?GGC?AAC 1708Asn?Glu?His?Lys?Gly?Lys?Lys?Ile?Gly?Gln?Val?Lys?Ser?His?Gly?Asn

510 515 520TTC?ACC?TTC?ATG?CGT?CTC?TAT?GGC?GGT?GGC?CAC?ATG?GTC?CCG?ATG?GAC 1756Phe?Thr?Phe?Met?Arg?Leu?Tyr?Gly?Gly?Gly?His?Met?Val?Pro?Met?Asp

The data of 525 530 535CAA CCC GAG TCG AGT CTT GAA TTC TTC AAC CGC TGG TTG GGA GGT GAA 1804Gln Pro Glu Ser Ser Leu Glu Phe Phe Asn Arg Trp Leu Gly Gly Glu540,545 550 555TGG TTT TAA AGACGTGCTA TCACCGCATA TAGACTTTCC GGTCATTTCG GTGACACTGC 1863Trp PheAGATATGTTT CTTAACGATA GTTTGAGGAT GCTTGTCAAT GCCCACTAAT CCCGAGCCTT 1923ATGTTACATG GTATCTATGA GTTTGTCATT ATAGTGCATT ATGCATTTGT ACTCCGTACG 1983AGAATGAATC AGCGGCCGC, 2002 (2) SEQ ID NO:4: (i) sequence signature:

(A) length: 557 amino acid

(B) type: amino acid

(C) chain: strand

(A) biology: aspergillus niger (xi) sequence description: SEQ ID NO:4:Met Arg Val Leu Pro Ala Ala Met Leu Val Gly Ala Gly Thr Ala Ala1 5 10 15Val Pro Pro Phe Gln Gln Val Leu Gly Gly Asn Gly Ala Lys His Gly

20 25 30Ala?Asp?His?Ala?Ala?Glu?Val?Pro?Ala?Asp?His?Ser?Ala?Asp?Gly?Phe

35 40 45Ser?Lys?Pro?Leu?His?Ala?Phe?Gln?Glu?Glu?Leu?Lys?Ser?Leu?Ser?Asp

85 90 95Pro?Asp?His?His?Trp?Asp?His?Ile?Val?Arg?Gly?Ser?Asp?Val?Gln?Ser

100 105 110Val?Trp?Val?Thr?Gly?Glu?Asn?Gly?Glu?Lys?Glu?Arg?Glu?Val?Asp?Gly

115 120 125Lys?Leu?Glu?Ala?Tyr?Asp?Leu?Arg?Val?Lys?Lys?Thr?Asp?Pro?Ser?Ser

165 170 175Asp?Pro?Glu?Asn?Asp?Pro?Val?Val?Leu?Trp?Leu?Asn?Gly?G1y?Pro?Gly

180 185 190Cys?Ser?Ser?Leu?Thr?Gly?Leu?Phe?Met?Glu?Leu?Gly?Pro?Ser?Ser?Ile

195 200 205Asn?Lys?Lys?Ile?Gln?Pro?Val?Tyr?Asn?Asp?Tyr?Ala?Trp?Asn?Ser?Asn

245 250 255Ala?Leu?Leu?Thr?Leu?Phe?Phe?Lys?Gln?Phe?Pro?Glu?Tyr?Ala?Lys?Gln

260 265 270Asp?Phe?His?Ile?Ala?Gly?Glu?Ser?Tyr?Ala?Gly?His?Tyr?Ile?Pro?Val

275 280 285Phe?Ala?Ser?Glu?Ile?Leu?Ser?His?Lys?Lys?Arg?Asn?Ile?Asn?Leu?Gln

290 295 300Ser?Val?Leu?Ile?Gly?Asn?Gly?Leu?Thr?Asp?Gly?Leu?Thr?Gln?Tyr?Glu305 310 315 320Tyr?Tyr?Arg?Pro?Met?Ala?Cys?Gly?Asp?Gly?Gly?Tyr?Pro?Ala?Val?Leu

325 330 335Asp?Glu?Gly?Ser?Cys?Gln?Ala?Met?Asp?Asn?Ala?Leu?Pro?Arg?Cys?Gln

340 345 350Ser?Met?Ile?Glu?Ser?Cys?Tyr?Ser?Ser?Glu?Ser?Ala?Trp?Val?Cys?Val

355 360 365Pro?Ala?Ser?Ile?Tyr?Cys?Asn?Asn?Ala?Leu?Leu?Ala?Pro?Tyr?Gln?Arg

405 410 415Thr?Glu?Val?Ile?Glu?Ala?Val?Gly?Ala?Glu?Val?Asn?Gly?Tyr?Asp?Ser

420 425 430Cys?Asn?Phe?Asp?Ile?Asn?Arg?Asn?Phe?Leu?Phe?His?Gly?Asp?Trp?Met

435 440 445Lys?Pro?Tyr?His?Arg?Leu?Val?Pro?Gly?Leu?Leu?Glu?Gln?Ile?Pro?Val

485 490 495Ser?Ala?Lys?Leu?Glu?Asp?Leu?Val?Val?Val?Glu?Asn?Glu?His?Lys?Gly

500 505 510Lys?Lys?Ile?Gly?Gln?Val?Lys?Ser?His?Gly?Asn?Phe?Thr?Phe?Met?Arg

515 520 525Leu?Tyr?Gly?Gly?Gly?His?Met?Val?Pro?Met?Asp?Gln?Pro?Glu?Ser?Ser

530 535 540Leu?Glu?Phe?Phe?Asn?Arg?Trp?Leu?Gly?Gly?Glu?Trp?Phe545 550 555

Claims

1. isolating carboxypeptidase of aspergillus niger Y.

2. isolating carboxypeptidase y, it by under very rigorous condition with the nucleic acid sequence encoding of SEQ IDNO:1 hybridization.

3. according to the carboxypeptidase y of claim 2, wherein nucleotide sequence derives from Aspergillus, fusarium, Penicillium, Humicola, Trichoderma, Scytalidium, myceliophthora or careless Rhizopus.

4. according to the carboxypeptidase y of claim 3, wherein nucleotide sequence derives from Aspergillus.

5. according to the carboxypeptidase y of claim 2, it has aminoacid sequence SEQ IDNO:2.

6. isolating carboxypeptidase y, it by under very rigorous condition with the nucleic acid sequence encoding of SEQ IDNO:3 hybridization.

7. according to the carboxypeptidase y of claim 6, wherein nucleotide sequence derives from Aspergillus, fusarium, Penicillium, Humicola, Trichoderma, Scytalidium, myceliophthora or careless Rhizopus.

8. according to the carboxypeptidase y of claim 7, wherein nucleotide sequence derives from Aspergillus.

9. carboxypeptidase y according to claim 6, it has aminoacid sequence SEQ IDNO:4.

10. according to the carboxypeptidase y of claim 1, it is by the nucleic acid sequence encoding among the pDSY23 that is included among the intestinal bacteria NRRL B-21326.